Clarifying Optimal Sodium InTake In Cardiovasular and Kidney (COSTICK) Diseases: a study protocol for two randomised controlled trials

Background: While low sodium intake (<2.3g/day) is recommended for all, there is uncertainty about feasibility and net cardiovascular effects. In COSTICK, we evaluated the effects of a dietary counselling intervention (reduced sodium intake) on intermediate cardiorenal outcomes in patients with (STICK) and without (COSIP) mild/moderate kidney disease. Methods: This is a protocol for two phase IIb randomised, two-group, parallel, open-label, controlled, single centre trials. Participants were aged >40 years with stable blood pressure, unchanged anti-hypertensive medications, willing to modify diet and provided written informed consent. Participants were excluded for abnormal sodium handling, heart failure, high dose diuretics, immunosuppression, pregnancy/lactation, postural hypotension, cognitive impairment, high or low body mass index (BMI) or inclusion in another trial. STICK participants had estimated glomerular filtration rate (eGFR) 30-60ml/min/1.73m 2 and were excluded for acute kidney Injury, rapidly declining eGFR; known glomerular disease or current use of non-steroidal anti-inflammatory drugs. For COSIP, participants were excluded for known kidney or cardiovascular disease. Participants were randomized to usual care only (healthy eating) or an additional sodium lowering intervention (target <100mmol/day) through specific counseling (sodium use in foods, fresh over processed foods, sodium content of foods and eating outside of home). In STICK the primary outcome is change in 24-hour urinary creatinine clearance. In COSIP, the primary outcome is change in five biomarkers (renin, aldosterone, high sensitivity troponin T, pro-B-type natriuretic peptide and C-reactive protein). Our primary report (COSTICK), reports six biomarker outcome measures in the entire population at 2 years follow-up. Discussion: These Phase II trials will explore uncertainty about low sodium intake and cardiovascular and kidney biomarkers, and help determine the feasibility of low sodium intake. Trial results will also provide preliminary information to guide a future definitive clinical trial, if indicated. Trial registration: STICK: ClinicalTrials.gov NCT02738736 (04/04/2016); COSIP: ClinicalTrials.gov NCT02458248 (15/05/2016)


Introduction
Effective, simple, inexpensive interventions are essential for population-based strategies to impact blood pressure (BP), which may impact the global burden of cardiovascular disease (CVD) and chronic kidney disease (CKD) 1 . Low sodium intake (<2.3g/day) is recommended for adults 2 , with specific emphasis on individuals with hypertension, CVD and CKD. In populations with hypertension (or pre-hypertension) the evidence supporting low sodium intake (compared to moderate intake, 2.3-4.6g/day) is based on Phase II trials reporting a reduction in BP 3-6 .
Meta-analyses of observational studies estimate a mean BP reduction of 10/5mmHg per 2.3g (100mmol) reduction in daily sodium intake 7 and meta-analysis of clinical trials estimate that reducing from moderate to low sodium intake reduced BP by 5.0/2.7mmHg 8 . It is assumed that all BP reductions will translate into reductions in CVD and CKD progression. However, there is also evidence of activation of the renin-angiotensin-aldosterone system (RAAS) with low sodium intake in the short-term 9 and with persistent effects 10,11 (e.g. Yanomami tribes in northwestern Brazil), and prospective cohort studies report a J-shaped association of sodium intake with cardiovascular health 1,12 . There have also been inconsistent reports about the association between sodium intake and biomarkers, including inflammatory biomarkers (C-reactive protein (CRP) and interleukin 6 (IL-6)), markers of cardiac injury/strain (high sensitivity troponin T (hsTnT) and pro B-type natriuretic peptide (proBNP) and of salt sensitivity (uromodulin) [13][14][15][16] .
For patients with CKD, no moderate or large-scale clinical trials have evaluated the effect of sustained low sodium intake on renal outcomes. Our systematic review of the association between dietary sodium intake/excretion and renal function showed that there is evidence of an association between high sodium intake, but not moderate intake, and glomerular filtration rate (GFR) decline among participants with CKD 17 . Subsequent, analyses of additional observational studies reported no association between urinary sodium excretion and renal outcomes [18][19][20] . A prospective cohort study from Korea reported a U-shaped association between sodium intake and CKD incidence 21 . A further meta-analysis of clinical trials of dietary sodium reduction in CKD included 11 trials (n=738) and reported that moderate sodium restriction reduced blood pressure and urinary protein excretion 22 .
These competing observations have cast considerable uncertainty about the optimal level of sodium intake, reflected in differences in guidelines and recommendations 23 . Therefore, the sustained effects of long-term low-sodium intake on biomarkers of cardiovascular and kidney function is a critical next step, necessary to inform the conduct of future definitive randomized controlled trials.

Protocol
Study aim and design Both trials were designed to explore the effects of an educational intervention to reduce sodium intake targeting low levels, compared to usual care. The Sodium In Take In Chronic Kidney Disease (STICK) clinical trial was designed to explore the effect of the intervention in adults with non-severe CKD on decline in renal function over two years. The Clarifying Optimal Sodium Intake in Populations (COSIP) clinical trial was designed to explore the effect of the intervention in adults without cardiovascular disease on a panel of cardiovascular biomarkers, over two years.
Both trials are Phase IIb randomised, two-group, parallel, open-label, controlled trials conducted at a single-centre. Blinding of participants and the research dietitian was not possible for practical reasons. Participants were recruited from hospital-based outpatient clinics or attending general practitioners in the catchment area of a tertiary referral hospital in Galway, Ireland. Potentially eligible participants were invited to participate and provided written informed consent before entering a screening period, consisting of an abbreviated food frequency questionnaire (FFQ; see extended data 24 ), medical history, laboratory investigations, baseline physical measurements (height, weight, body mass index (BMI), hip and waist circumference) and blood pressure (BP). Office BP was measured using a calibrated, automated oscillometric device and followed by a 24-hour ambulatory blood pressure monitor (ABPM) using the Spacelabs ABP 90217 device, where BP was measured every 30 minutes between 7am and 10pm, and every 60 minutes between 10pm and 7am. Given the complementary nature of both trials, participants were screened for both trials simultaneously, but only deemed eligible and randomized into STICK or COSIP. Rescreening of participants who failed the screening visit due to a blood pressure result or medication change was permitted after a minimum of three months.
Eligibility criteria for both trials Inclusion criteria for both trials included age >40 years, stable blood pressure, unchanged anti-hypertensive medications, willingness to modify dietary intake and written informed consent (Table 1). Initially, we also required dietary assessed sodium intake of >2.3g/day, but removed this after the Vanguard phase due to weak correlation between the dietary sodium assessment and baseline 24 hour urine sodium collection. Exclusion

Amendments from Version 1
Our protocol manuscript was revised in response to the comments of two peer reviewers. We revised a number of references throughout the manuscript to provide additional clarifications and updates on key literature, including additional details on the activation of renin in Yanomami tribes. We provided additional clarity on the scheduling of patients for randomisation visits -some participants were randomised in advance of their visit to facilitate scheduling of patients. We also provide further detail for obtaining blood samples from participants and initial processing and storage and corrected an oversight that we measured direct renin in participants (rather than plasma renin activity). We have responded to other comments raised by the reviewers, but not modified the manuscript.
Any further responses from the reviewers can be found at the end of the article Eligibility criteria -STICK specific All STICK participants were required to have a stable estimated glomerular filtration rate (eGFR) of 30-60ml/min/1.73m 2 within three months of randomization. Participants were excluded for recent acute kidney Injury (AKI), rapidly declining eGFR 25 , known glomerular disease or current use of non-steroidal anti-inflammatory drugs (NSAID).
Eligibility criteria -COSIP specific For COSIP, participants were excluded for known CKD or CVD.

Randomisation
Once confirmed eligible, participant details were entered into a trial-specific, secure, web-based randomisation service. In STICK, we used a minimization strategy (including previous attendance at a dietitian, diabetes, hypertension and strata of eGFR [30-39, 40-49 and 50-60ml/min/1.73m 2 ]) to support computergenerated block randomization with 1:1 assignment to intervention or usual care. In COSIP, we used computer-generated block randomization to 1:1 assign participants to intervention or usual care. Variable block sizes were used to minimize the occurrence of chance imbalances and to preserve allocation concealment. After confirmation of eligibility, participants may have been randomised in advance to facilitate the scheduling of individuals to attend distinct clinics for either the intervention or usual care arms.

Study interventions
Usual care: All participants received a one-to-one, dietitiandeveloped healthy eating guidance session 26 , administered by a trained member of the research team, over 15 minutes following randomization, in addition to written materials emphasizing key messages 27 . Telephone contact was made at months nine and 15 to follow-up on key points on healthy eating (each 15 minutes) with a total contact time of 45 minutes over the course of the trials. Participants randomised to usual care did not meet with the dietitian nor did they receive focused recommendations on sodium intake (other than general guidance contained in 'Health Food for Life' 27 ).
Sodium reduction intervention: In addition to healthy eating guidance sessions, those randomised to the intervention received specific counseling on behavioural and environmental factors to promote reduction in sodium intake to a target of <100mmol/day (<2.3g/day). A research dietitian developed the specific components of the intervention, based on the trial of non-pharmacologic interventions in the elderly (TONE) trial 28 . A registered dietitian delivered the intervention using an operations manual to standardize delivery within and across participants. In circumstances where the research dietitian was unavailable, an appropriately trained delegate delivered the intervention. The intervention targeted: (i) reducing use of 'salt' during food preparation (encouraging the use of herbs and/or spices); (ii) reducing table 'salt' use; (iii) encouraging fresh food consumption over processed or canned foods; (iv) identification of sodium content in foods; (v) modifying the consumption of foods with high sodium content; and (vi) advice on eating outside of home. After determining health literacy, using the Newest Vital Sign UK (NVS-UK) 29 , participants were counselled and behaviour change techniques (goal setting, self-monitoring, frequent contact, feedback and re-enforcement, problem solving and motivational interviewing) were employed to reduce sodium intake by setting specific, measurable, achievable, realistic and timely (SMART) goals for the individual. Tools used included 24-hour dietary recall (analysed using Nutritics 30 ), three-day food diaries and sodium behavior checklists.
The intervention was delivered at all patient contacts up to and including T 7 (21 month) visit (Table 2) with approximately 225-255 minutes of in-person contact and 155 minutes of telephone contact over the course of the trials. Participants had in-person, one-to-one sessions with the study dietitian at randomization (T 0 ), vanguard (T V ), where relevant, and T 2 ; group sessions at T 1 and T 4 . In addition, each participant received five separate 15 min telephone calls (Week 1, Week 2, Week 4, Week 6 and Week 10) and 20 min telephone calls at T 3 , T 5 , T 6 and T 7 .

Follow-up schedule
To minimize participant inconvenience and burden, six or seven in-person visits were considered essential (screening, randomization (T 0 ), T v (first 20 participants for each trial), T 1 , T 2 , T 4 and T 8 ) and the remaining four follow-up visits were carried out by telephone (T 3 , T 5 , T 6 and T 7 ) (Table 3). Post-trial, participants will return to routine clinical care.

Data collection
The following data were collected using standardised case report forms (CRF): demographics, physical measurements (BP, height, weight, waist and hip circumference), medications, medical history, lifestyle factors, randomization allocation, diet, delivery of healthy eating guidance session, delivery of the sodium reduction intervention, clinical events, hospitalisations, adverse events and laboratory results (see extended data 24 ). Diet was measured using FFQ, food diaries and a questionnaire focused on sodium-specific behaviours (see extended data 24 ). Blood samples were drawn at the time of clinic attendance from participants in the seated position and using a tourniquet. For renin and aldosterone, an EDTA sample was drawn and spun at 2000g for 10minutes at 20 degrees Celsius within 30minutes of phlebotomy. Plasma was then frozen at -20 or -80 degrees Celsius until periodically transferred to our local laboratory using styrofoam boxes and cooler packs to ensure samples did not defrost. Laboratory samples were analysed at the Galway University Hospital laboratory using standardised storage, handling and analytical procedures. We measured serum sodium, potassium, creatinine, glycated haemoglobin (HbA1c), high-density lipoprotein (HDL), low-density lipoprotein (LDL), direct renin, aldosterone, high sensitivity troponin T (hsTnT), Pro B-type Natriuretic Peptide (proBNP), C-reactive protein (CRP), urine protein creatinine ratio (uPCR), 24 hour urine (sodium, potassium, creatinine and protein) and fasting morning urine (sodium, creatinine and protein). This included serum creatinine, measured using methodology traceable to an isotope dilution mass spectrometry (IDMS) reference measurement. Samples were transported to the laboratory within one hour of blood draw and analyses were conducted using standardized laboratory methods on the Roche Cobas® 8000 modular analyser series (Roche Diagnostics Limited, West Sussex, UK), except for HbA1c which was measured using capillary electrophoresis on the Sebia Capillarys 3 automated haemoglobin analyser using the Sebia HbA1c kit (2017/05). After additional consent was obtained, additional blood and urine samples, as well as PAXgene samples for RNA measurement, for biobanking were taken at T 0 , T 1 and T 8 . To ensure high quality data collection, staff received training on completing paperbased CRF and additional training on entering data into the study database, which included database integrity checks to ensure data consistency.

Study outcomes
In STICK, the primary outcome measure is change in 24-hour urinary creatinine clearance over two years. We adopt this approach, rather than eGFR, to improve reliability and validity of the primary outcome measure. In COSIP, the primary outcome measure is change in cardiovascular biomarkers (renin, aldosterone, hsTnT, proBNP and CRP) over two years. For COSTICK, the primary outcome measures are creatinine clearance,

Safety reporting
An adverse event is defined as any untoward medical occurrence, including an exacerbation of a pre-existing condition or abnormal laboratory finding, with or without a causal relationship with sodium intake. Participants were monitored for adverse events from informed consent through completion of the final study visit. All adverse events were captured on case report forms, entered into the clinical trial database and coded using the Medical Dictionary for Regulated Activities (MedDRA). Annual line listings of adverse events were reported to the ethics committee.

Withdrawal of intervention
The study intervention was withdrawn if: (i) symptomatic orthostatic hypotension with systolic BP ≤100mmHg; (ii) diagnosis of glomerular disease (as listed in exclusion criteria); (iii) diagnosis of ESRD, initiation of dialysis or renal transplantation; (iv) prescription of sodium bicarbonate therapy or treating physician recommended against sodium restriction; (v) pregnancy; or (vi) acquired new dietary requirement that precluded the intervention. These participants were encouraged to complete all remaining follow-up visits.

Sample size calculation
For STICK, we assumed a mean decline in creatinine clearance of 8±5ml/min/1.73m 2 over the trial period 33 and estimated a minimum clinically meaningful effect size of 25% relative reduction. Based on an alpha of 0.05 and power of 80%, a per group sample size of 99 participants was required. Assuming a dropout rate of 5% participants, a net crossover/non-adherence of 5% in favour of the control group, we required a total sample size of 224 participants. For COSIP, we based sample size on the ability to detect an effect size of 0.40 in the between-group difference in mean change scores of biomarkers (80% power and alpha 0.05), equating to a difference of 0.4 of the standard deviation of the change in biomarker. As the COSIP trial is considered exploratory, each biomarker is tested at significance threshold p<0.05. Assuming a dropout rate of 5% participants, a net crossover/non-adherence of 15%, we require a total sample size of 286 participants. Combining COSIP and STICK trials resulted in the ability to detect a smaller effect size.

Statistical analysis plan
Descriptive statistics will be used to describe baseline characteristics, the flow of trial participants and the amount of missing data for predictor and outcome variables. All losses to follow-up and dropouts will be accounted for and reasons documented. Participants randomised but who did not attend the randomisation visit will be included in analyses. Suitable transformations will be used if the underlying normality assumption is a concern and the bootstrap procedure and a comparison of the median change will be used if a suitable transformation cannot be made. All tests of significance will be two-sided using an alpha of 0.05 for statistical significance and will not be adjusted for multiple testing due to the exploratory nature of the trials. The primary analysis will use the intention-to-treat dataset for all outcomes. A secondary per-protocol analysis will also be carried out, with post randomisation exclusions due to ineligibility, non-compliance, loss to follow-up or missing data.
The primary analysis will consist of fitting a separate linear, multivariable analysis to each of the six outcomes: renin, aldosterone, creatinine clearance, pro-BNP, troponin, and CRP, and secondary outcome measure of 24-hour ABPM. We will review data for implausible values and exclude extreme changes (e.g. change in renin or aldosterone impacted by change in medications). Corresponding baseline measurements of each biomarker will be included as covariates in the model, along with a factor variable for the sub-study in which each patient was recruited, age, sex and whether the participant had attended a dietitian in the 6 months previous to baseline (incident vs prevalent participant). To ensure estimates are more robust to potential effects of missing data at 24-month follow-up, an inverse probability weighting will be applied in all regression analyses to account for the differing propensity of patients with certain baseline characteristics to be included in the final analysis. All covariates included in the regression model will be included in the missingness model, including the other 5 primary endpoints.
Reporting of primary analysis results will consist of tabulations of the unadjusted and adjusted mean differences at 24 months between intervention and control groups for each biomarker, with confidence intervals of these parameter estimates and p-values, along with descriptive unadjusted mean differences at 24 months between intervention and control groups. A forest plot of the standardised, adjusted mean difference for all 6 endpoints will also be generated. Statistical tests of interaction (Wald) will be used for all subgroup analyses. A sensitivity analysis will be performed to assess the effect of participant reported compliance on treatment effect. Due to the duration of this trial, we expect to have a complete dataset for covariate and outcome data for 90% of participants. Complete case analysis will be used to handle missing data. A sensitivity analysis with multiple imputation will also be carried out if there is >10% missing data for covariates.

Data monitoring
A data and safety monitoring board (DSMB), chaired by a clinician scientist with experience in clinical trials, reviewed the progress of the trials and safety data when 50% of participants completed one-year follow-up, when the DSMB recommended that the trials proceed. An independent study monitor reviewed the integrity of study data at multiple timepoints throughout the trials and ensured that study procedures were compliant with the International Conference on Harmonisation Good Clinical Practice (ICH-GCP).

Study administration
These trials are single centre studies with all activities conducted and coordinated at the HRB Clinical Research Facility Galway (HRB-CRFG). The investigators did not implement any deviation from, or changes to, the trial protocols without review and documented approval from the research ethics committee. All participants provided written informed consent before any study specific activities were completed, and all data was handled with strict privacy and electronic data security standards, using unique subject identifiers to prevent unauthorised identification of research participants. Pseudononymised data were entered into a study-specific, secure, password-protected clinical trial database with an audit trail.

Dissemination of information
The results of the trials will be presented at scientific meetings (local, national and international), patient-oriented meetings and published in peer-reviewed publications. The HRB-CRFG and investigators have the ownership of all data and results collected during the trials and have full rights to publication, without restriction. For all publications and presentations from this work, authors will be required to make substantial contributions to (i) concept or design; acquisition, analysis or interpretation of data; (ii) drafting or critically revising manuscripts and (iii) give final approval of the version to be published, in line with the International Committee of Medical Journal Editors guidelines for authorship. The datasets will not be made publicly available, instead they will be maintained at HRB-CRFG and the investigators must review and approve all requests for access to the data.

Study status
Recruitment of both clinical trials is closed and the final visits of all patients have taken place. Data cleaning is ongoing, ahead of database lock, statistical analyses and trial reporting.

Discussion
Clinical trials that challenge current guidelines are difficult but are of considerable importance, with major implications for management of patients and populations. For those consuming high sodium diets (>200mmol/day), there is convincing evidence of negative associations including reduced eGFR 17,34 , increased proteinuria 17,35,36 and adverse associations with blood pressure and CVD. However, there is major uncertainty about whether low sodium intake is associated with more benefit than harm, which needs to be interrogated with clinical trials. Dietary trials over medium to long durations are particularly challenging, but data from rigorously conducted trials are necessary to appropriately inform guidelines. These trials will help determine the feasibility of sodium restriction to current guideline levels and determine if this reduction in intake is sustainable. The results will provide preliminary information, to guide future definitive clinical trials, if indicated.
Dietary sodium intake is modifiable, as evidenced by numerous clinical trials of standardized educational interventions that successfully reduced dietary sodium intake to low intake range over the short-term 28,37 , including a small, short duration feasibility clinical trial in CKD 38 . It is clear from such trials that a sodium reduction intervention strategy will reduce sodium intake, but the amount and sustainability of the reduction is less clear. We designed our intervention based on the TONE trial 28 , targeting non-discretionary sources of sodium intake. After the Vanguard phase (first 20 participants in each trial) was completed, we observed that mean 24 hour urine sodium excretion reduced from 163(62)mmol/day to 125(53)mmol/day, suggesting that the intervention was effective in lowering sodium intake.
Our trials have a number of limitations. First, the trials are open-label as it wasn't practical to blind participants, investigators or study staff to the intervention. However, to minimize the risk of bias we will perform blinded outcome assessment including blinding of the trial statistician. Second, participants in the usual care arm may receive non-study recommendations on sodium reduction, reducing the likelihood of observing the effect from our intervention, biasing toward the null. To minimise this risk of bias, treating clinicians were asked not to explicitly discuss sodium reduction with participants unless strongly indicated and the trial intervention was to be withdrawn. Similarly, as contamination is a threat to trial validity, only participants randomized to the intervention met with the study dietitian, participants were instructed not to discuss the educational sessions with other participants and participants were invited to attend follow-up visits on days based on study allocation. Third, there is a risk of volunteer bias, as participants may differ from non-participants, impacting trial generalizability. We will measure the potential effect of this bias by comparing non-participants and participants. Fourth, the primary outcome measures are changes in biomarkers rather than hard clinical endpoints, which would require a trial of longer duration (to accumulate a sufficient number of clinical events) to demonstrate a treatment effect. The findings of our trials can be used to inform future, larger, multicenter trials. Fifth, individuals with CKD may have previously consulted with a dietitian and received advice on lowering sodium intake, limiting their 'responsiveness' to our intervention. Therefore, we included previous dietitian review in the minimization strategy for randomization in STICK; in addition, we believe that the trial intervention is more intensive than standard care. Finally, there is a risk of non-compliance with the intervention and a reliance on participant self-reported adherence as this is a behavioural intervention trial. To maximise compliance, we: (i) self-selected participants most likely to adhere to a behavioural intervention; (ii) implemented standardised telephone follow-up to provide additional advice and support, answer questions, reinforce key messages and remind participants about visits; and (iii) provided written materials on the study interventions. We also completed multiple 24-hour urine collections for sodium to measure objectively measure adherence, although these collections also have limitations 39 .
Ethics approval and consent to participate: The STICK trial was conducted in accordance with (ICH-GCP) 40  Reviewer Expertise: Cardiology. Epidemiology.

Introduction
The authors state 'However, there is also evidence of activation of the renin-angiotensin aldosterone system (RAAS) with low sodium intake from short-term studies (mostly <6 months)' but in fact, this RAAS activation is also well documented in situations of low sodium intake for a long time, as occurs among Yanomami tribes in northwestern Brazil.

Eligibility Criteria
Why was the use of azathioprine or mycophenolate use exclusion criteria? Should not the use of SGLT2 inhibitor be an exclusion criterion?
○ What is the reference for the upper limit for the dose of diuretic to be considered high?

○
The meaning of the statement 'Eligible participants may have been randomised prior to attending the randomisation visit to facilitate the scheduling of separate intervention and usual ○ care clinics' is not clear.

Intervention
Randomization should be to similar diets, with the only difference being the reduction in the sodium intake, as a low-sodium diet may differ in other dietary factors, such as potassium intake, which has also been associated with cardiorenal endpoints.

Data Collection
There is not enough information about the collection and analysis of laboratory variables, especially for hormones and enzymes, which depend on body position and time of day to interpret the results.

○
In the Data Collection section, there is the statement 'After additional consent was obtained, additional blood and urine samples for PAXgene samples for RNA measurement and for biobanking were taken at T0, T1 and T8.'. with no prior mention of RNA analysis, details on methodology or type of genetic analysis to be performed. Please clarify.

Study Outcomes
As there is some evidence that sodium restriction also has adverse effects of activating the renin angiotensin aldosterone system, increasing catecholamines, and adversely affecting insulin sensitivity and lipids, the inclusion of some measure of insulin sensitivity as an outcome would be desirable.

Sample Size Calculation
It is not clearly explained, and the calculation report does not support the minimal number of participants that the authors describe. If the calculation has not been done prior to the randomization, a power calculation may be an acceptable option due to the exploratory nature of the trial.

Statistical Analysis Plan
Considering that a low-sodium diet may have different effects according to the subgroup of individuals, I suggest an exploratory analysis of subgroups, especially for diabetic patients and patients using angiotensin converting enzyme inhibitors or angiotensin receptor blockers. There is evidence that a low-sodium diet is associated with increased cardiovascular and all-cause mortality in diabetic patients, due to greater activation of the sympathetic nervous system and RAAS, in addition to greater insulin resistance.

Are sufficient details of the methods provided to allow replication by others? Partly
Are the datasets clearly presented in a useable and accessible format?
Not applicable overall dietary advice. Our participants were randomised to receive specific counselling targeting sodium intake or not (usual care).

Comment:
There is not enough information about the collection and analysis of laboratory variables, especially for hormones and enzymes, which depend on body position and time of day to interpret the results. Response: We provided additional detail on the collection of blood samples and the protocol used

Comment:
In the Data Collection section, there is the statement 'After additional consent was obtained, additional blood and urine samples for PAXgene samples for RNA measurement and for biobanking were taken at T0, T1 and T8.'. with no prior mention of RNA analysis, details on methodology or type of genetic analysis to be performed. Please clarify. Response: These samples were obtained and stored for potential future use and are not part of the primary trial report.

Comment:
As there is some evidence that sodium restriction also has adverse effects of activating the renin angiotensin aldosterone system, increasing catecholamines, and adversely affecting insulin sensitivity and lipids, the inclusion of some measure of insulin sensitivity as an outcome would be desirable. Response: We designed the trials around the outcomes of cardiovascular and renal function. We did not complete measurements of catecholamines or insulin sensitivity during the trial. We thank the reviewer for raising these issues, which we will consider exploring in future posthoc work with the biobanked samples.

Comment:
It is not clearly explained, and the calculation report does not support the minimal number of participants that the authors describe. If the calculation has not been done prior to the randomization, a power calculation may be an acceptable option due to the exploratory nature of the trial. Response: The rationale and underlying assumptions (alpha, power, expected baseline change in biomarker, clinically meaningful effect size, dropout and potential for crossover/nonadherence) are provided for the STICK and COSIP trials.

Comment:
Considering that a low-sodium diet may have different effects according to the subgroup of individuals, I suggest an exploratory analysis of subgroups, especially for diabetic patients and patients using angiotensin converting enzyme inhibitors or angiotensin receptor blockers. There is evidence that a low-sodium diet is associated with increased cardiovascular and all-cause mortality in diabetic patients, due to greater activation of the sympathetic nervous system and RAAS, in addition to greater insulin resistance. Response: We thank the reviewer for this comment. The primary report of the trial results will include subgroup analyses and we will distinguish between pre-specified and post-hoc subgroup analyses. are no studies to support beneficial health effects of such a reduction. Furthermore, the COSTICK study includes an outcome, which is not just a "biochemical" outcome, but a true morbidity outcome, as it reflects the effect on the renal function over a 2-year period. In contrast, the COSIP study "only" reflects cardiovascular biomarkers, but some of them have not been invetigated previously and may contribute to establish whether a low sodium diet changes the disposition (for instance increases or decreases hsTnT and proBNP) to true cardiovascular events. Although it must be expected that aldosterone and renin will increase during the low sodium diet according to reference 9 and a new study (DOI:https://doi.org/10.1016/j.eclinm.2021.100750) 1 , it is worth to investigate this effect over a 2-year period.

Study intervention
The diet counselling is more intense in the intervention group, which leaves the question whether there will be other dietary differences between the two groups than differences in sodium intake (Table 2). Such interactions may complicate the final interpretation of the results. The many 24-h urine measurements (Table 3) eliminate one of the previous issues of conflict (the method of sodium intake measurement), which has resulted in reservations on the interpretation of outcomes in previous studies.

Discussion
The study is well designed, the statistical procedures are appropriately described, and the level of sodium reduction is controlled by multiple 24-h urine measurements. These preconditions should ensure solid conclusions. The authors state that "These trials will help determine the feasibility of sodium restriction to current guideline levels and determine if this reduction in intake is sustainable." This is an The obvious explanation for this change in duration strategy from 36 months to 1 month is that the sodium reduction and blood pressure effects achieved in the longer-term HPT and TOHP studies were small. This leaves some questions. Why is the official policy to reduce sodium intake to below 2.3 g, if this strategy is not feasible for more than 1 month, even under controlled circumstances? And why did NHLBI support a short-term sodium reduction study, knowing that a sodium reduction strategy to below 2.3 g was not feasible for a longer period? In this context it will be exciting to learn whether the present studies will show that such a strategy is possible or not, and it will be important to interpret the outcomes in relation to the achieved sodium reduction. If the goal to reduce sodium intake to below 2.3 g is not achieved, it is reasonable to raise the question why it is difficult to get below that level. Is it because the normal human neuro-endocrinological physiology counteracts a sodium intake below 2.3 g, simply because the net-effect of such a low sodium intake is harmful, as indicated by the activation of RAAS at sodium intakes below 2.3 g? In this context it is predictable that renin and aldosterone will only increase significantly, if the goal to reduce sodium intake below 2,3 is fulfilled. From a scientific and evidence-based point of view the outcomes of the COSTICK and COSIP studies will be exciting. If the studies show the "right" results and support the present sodium reduction policy, the studies will be well-received by the health authorities. But, if the results "wrongly" are in opposition to the existing sodium reduction policy, they will have no impact, because the political decision makers in the health organizations for decades have explained away studies, which do not confirm the official policy.
does not support that view, as this analysis does not investigate the independent association of a low sodium intake with health outcomes, but only the association of a high sodium intake (reference 10). Response: Reference 10 has been removed.

Comment:
The authors give a reference to their systematic review of the association between dietary sodium intake/excretion and renal function. It would be helpful to mention this reference, when mentioned the first time in the introduction, i.e. move reference 32 to reference 16. In this context the authors may benefit from consulting two additional references: Kidney International 2018; 93: 921-931 and Kidney International 2018; 93: 776-778, to further explore the known evidence of the association between sodium intake and renal function.

Response:
We have altered our references to include our systematic review at an early stage and include the additional references on the association between sodium intake and renal function.

Comment:
Reference 20 is not a guideline or a recommendation, but a commentary, which modifies an Institute of Medicine report commissioned by the Centre of Disease Control and Prevention to review and assess potential benefits and adverse outcomes of reducing sodium intake in the population (Institute of Medicine. 2013. Sodium Intake in Populations: Assessment of Evidence. Washington, DC: The National Academies Press, 2013). Likewise, reference 21 is also an opinion paper. It would be more precise to mention the original IOM report and to mention that there is a discrepancy between the inconclusive outcome of the original commissioned independent report intended to form the basis for the guidelines and the final definitive conclusion of the guidelines (reference: Dietary Guidelines for the Americans https://health.gov/dietaryguidelines/2015/guidelines/ (2015-2020).

Response:
We have updated our references in line with the recommendation of the reviewer.

Comment:
The purpose was to explore the effects of an educational intervention to reduce sodium intake targeting low levels. This is the right question, because the recommendations are not just to lower sodium intake, but to lower sodium intake to low levels below 2.3 g, the problem being that there are no studies to support beneficial health effects of such a reduction. Furthermore, the COSTICK study includes an outcome, which is not just a "biochemical" outcome, but a true morbidity outcome, as it reflects the effect on the renal function over a 2-year period. In contrast, the COSIP study "only" reflects cardiovascular biomarkers, but some of them have not been investigated previously and may contribute to establish whether a low sodium diet changes the disposition (for instance increases or decreases hsTnT and proBNP) to true cardiovascular events. Although it must be expected that aldosterone and renin will increase during the low sodium diet according to reference 9 and a new study (DOI:https://doi.org/10.1016/j.eclinm.2021.100750) 1 , it is worth to investigate this effect over a 2-year period. Response: We thank the reviewer for this comment.

Comment:
The diet counselling is more intense in the intervention group, which leaves the question whether there will be other dietary differences between the two groups than differences in sodium intake (Table 2). Such interactions may complicate the final interpretation of the results. The many 24-h urine measurements (Table 3) eliminate one of the previous issues of conflict (the method of sodium intake measurement), which has resulted in reservations on the interpretation of outcomes in previous studies. Response: We thank the reviewer for this comment and agree that it is possible that additional dietary differences may occur as approaches to alter diet to reduce sodium intake may alter the intake of other nutrients, particularly potassium. As such, we are measuring 24hour urine potassium excretion throughout the trial.

Comment:
The study is well designed, the statistical procedures are appropriately described, and the level of sodium reduction is controlled by multiple 24-h urine measurements. These preconditions should ensure solid conclusions. The authors state that "These trials will help determine the feasibility of sodium restriction to current guideline levels and determine if this reduction in intake is sustainable." This is an important point, The obvious explanation for this change in duration strategy from 36 months to 1 month is that the sodium reduction and blood pressure effects achieved in the longer-term HPT and TOHP studies were small. This leaves some questions. Why is the official policy to reduce sodium intake to below 2.3 g, if this strategy is not feasible for more than 1 month, even under controlled circumstances? And why did NHLBI support a shortterm sodium reduction study, knowing that a sodium reduction strategy to below 2.3 g was not feasible for a longer period? In this context it will be exciting to learn whether the present studies will show that such a strategy is possible or not, and it will be important to interpret the outcomes in relation to the achieved sodium reduction. If the goal to reduce sodium intake to below 2.3 g is not achieved, it is reasonable to raise the question why it is difficult to get below that level. Is it because the normal human neuro-endocrinological physiology counteracts a sodium intake below 2.3 g, simply because the net-effect of such a low sodium intake is harmful, as indicated by the activation of RAAS at sodium intakes below 2.3 g? In this context it is predictable that renin and aldosterone will only increase significantly, if the goal to reduce sodium intake below 2,3 is fulfilled. Response: We thank the reviewer for this comment and look forward to exploring such issues in the analysis and dissemination of our trial findings.
Comment: From a scientific and evidence-based point of view the outcomes of the COSTICK and COSIP studies will be exciting. If the studies show the "right" results and support the present sodium reduction policy, the studies will be well-received by the health authorities. But, if the results "wrongly" are in opposition to the existing sodium reduction policy, they will have no impact, because the political decision makers in the health organizations for decades have explained away studies, which do not confirm the official policy. Response: We thank the reviewer for this comment.